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RESEARCH ARTICLE
Zika Virus Infection as a Cause of Congenital
Brain Abnormalities and Guillain–Barre
Syndrome: Systematic Review
Fabienne Krauer1, Maurane Riesen1, Ludovic Reveiz2, Olufemi T. Oladapo3,
Ruth Martınez-Vega4, Teegwende V. Porgo3,5, Anina Haefliger1, Nathalie J. Broutet3,
Nicola Low1*, WHO Zika Causality Working Group¶
1 Institute of Social and Preventive Medicine, University of Bern, Switzerland, 2 Pan American Health
Organization, Washington DC, United States of America, 3 UNDP/UNFPA/UNICEF/WHO/World Bank
Special Programme of Research, Development and Research Training in Human Reproduction (HRP),
Department of Reproductive Health and Research, World Health Organization, Geneva, Switzerland,
4 Escuela de Microbiologia, Universidad Industrial de Santander, Santander, Colombia, 5 Department of
Social and Preventative Medicine, Laval University, Quebec, Canada
¶ Membership of the WHO Zika Causality Working Group is provided in the Acknowledgments.
Abstract
Background
The World Health Organization (WHO) stated in March 2016 that there was scientific con-
sensus that the mosquito-borne Zika virus was a cause of the neurological disorder Guil-
lain–Barre syndrome (GBS) and of microcephaly and other congenital brain abnormalities
based on rapid evidence assessments. Decisions about causality require systematic
assessment to guide public health actions. The objectives of this study were to update and
reassess the evidence for causality through a rapid and systematic review about links
between Zika virus infection and (a) congenital brain abnormalities, including microcephaly,
in the foetuses and offspring of pregnant women and (b) GBS in any population, and to
describe the process and outcomes of an expert assessment of the evidence about
causality.
Methods and Findings
The study had three linked components. First, in February 2016, we developed a causality
framework that defined questions about the relationship between Zika virus infection and
each of the two clinical outcomes in ten dimensions: temporality, biological plausibility,
strength of association, alternative explanations, cessation, dose–response relationship,
animal experiments, analogy, specificity, and consistency. Second, we did a systematic
review (protocol number CRD42016036693). We searched multiple online sources up to
May 30, 2016 to find studies that directly addressed either outcome and any causality
dimension, used methods to expedite study selection, data extraction, and quality assess-
ment, and summarised evidence descriptively. Third, WHO convened a multidisciplinary
panel of experts who assessed the review findings and reached consensus statements to
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 1 / 27
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OPENACCESS
Citation: Krauer F, Riesen M, Reveiz L, Oladapo
OT, Martınez-Vega R, Porgo TV, et al. (2017) Zika
Virus Infection as a Cause of Congenital Brain
Abnormalities and Guillain–Barre Syndrome:
Systematic Review. PLoS Med 14(1): e1002203.
doi:10.1371/journal.pmed.1002203
Academic Editor: Lorenz von Seidlein, Mahidol-
Oxford Tropical Medicine Research Unit,
THAILAND
Received: August 25, 2016
Accepted: November 16, 2016
Published: January 3, 2017
Copyright: © 2017 Krauer et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data files
are available from the Bern Open Repository and
Information System (BORIS) (http://boris.unibe.ch/
90317/).
Funding: The review was funded by the World
Health Organization (www.who.int, contract
numbers 2016/611294-0 and 2016/630126-0
awarded to NL) and the Swiss National Science
Foundation (www.snf.ch, SNSF special action fund
and project grant 320030_170069 awarded to NL).
source: https://doi.org/10.7892/boris.93383 | downloaded: 8.11.2020
update the WHO position on causality. We found 1,091 unique items up to May 30, 2016.
For congenital brain abnormalities, including microcephaly, we included 72 items; for eight
of ten causality dimensions (all except dose–response relationship and specificity), we
found that more than half the relevant studies supported a causal association with Zika virus
infection. For GBS, we included 36 items, of which more than half the relevant studies sup-
ported a causal association in seven of ten dimensions (all except dose–response relation-
ship, specificity, and animal experimental evidence). Articles identified nonsystematically
from May 30 to July 29, 2016 strengthened the review findings. The expert panel concluded
that (a) the most likely explanation of available evidence from outbreaks of Zika virus infec-
tion and clusters of microcephaly is that Zika virus infection during pregnancy is a cause of
congenital brain abnormalities including microcephaly, and (b) the most likely explanation of
available evidence from outbreaks of Zika virus infection and GBS is that Zika virus infection
is a trigger of GBS. The expert panel recognised that Zika virus alone may not be sufficient
to cause either congenital brain abnormalities or GBS but agreed that the evidence was suf-
ficient to recommend increased public health measures. Weaknesses are the limited
assessment of the role of dengue virus and other possible cofactors, the small number of
comparative epidemiological studies, and the difficulty in keeping the review up to date with
the pace of publication of new research.
Conclusions
Rapid and systematic reviews with frequent updating and open dissemination are now
needed both for appraisal of the evidence about Zika virus infection and for the next public
health threats that will emerge. This systematic review found sufficient evidence to say that
Zika virus is a cause of congenital abnormalities and is a trigger of GBS.
Author Summary
Why Was This Study Done?
• In 2015, the mosquito-borne Zika virus caused epidemics of a mild viral illness for the
first time in Brazil and then other countries in Latin America and the Caribbean.
• In mid to late 2015, clinicians in northeastern Brazil reported unexpected increases in
the numbers of babies born with abnormally small heads (microcephaly) and of adults
with Guillain–Barre syndrome (GBS), a paralytic condition triggered by certain
infections.
• In February 2016, the World Health Organization (WHO) declared a Public Health
Emergency of International Concern and called for research about the causal relation-
ship between Zika virus and congenital brain abnormalities, including microcephaly,
and GBS.
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 2 / 27
The following World Health Organization staff are
coauthors of the study: LR, OTO, and NJB. They
were involved in the study design, data
interpretation, decision to publish, and preparation
of the manuscript. The WHO Zika Causality
Working Group was involved in interpretation of
the data and the decision to publish.
Competing Interests: I have read the journal’s
policy and the authors of this manuscript have the
following competing interests: NL receives a
stipend as a specialty consulting editor for PLOS
Medicine and serves on the journal’s editorial
board. FK and MR received salary support from
WHO and/or SNSF to conduct the work. RMV was
a consultant of PAHO. All other authors have
declared that no competing interests exist.
Abbreviations: GBS, Guillain–Barre syndrome;
GRADE, Grading of Research Assessment
Development and Evaluation; MBP, myelin basic
protein; NPCs, neural progenitor cells; PAHO, Pan
American Health Organization; PRISMA, Preferred
Reporting Items for Systematic Reviews and Meta-
Analyses; RT-PCR, reverse transcriptase PCR;
WHO, World Health Organization.
What Did the Researchers Do and Find?
• We developed a causality framework for Zika virus and (a) congenital brain abnormali-
ties, and (b) GBS. For each outcome, we developed specific questions in ten different
dimensions of causality: temporality; biological plausibility; strength of association;
exclusion of alternative explanations; cessation; dose–response relationship; animal
experimental evidence; analogy; specificity; and consistency of findings.
• We did a systematic review of published and unpublished evidence up to May 30, 2016.
We summarised the evidence descriptively. A panel of experts assessed the findings and
reached a consensus about causality.
• For congenital brain abnormalities, we assessed 72 studies that addressed questions
in one or more causality dimensions. Reports of pregnancies affected by Zika virus
have come from countries with circulating Zika virus in the Americas, the Pacific
region, and West Africa. Clinical reports have documented Zika virus infection in
pregnant women followed by foetal abnormalities, particularly with infection in the
first trimester. These women did not have any other congenital infection or dengue
virus infection. The risk of congenital brain abnormalities could be around 50 times
higher in mothers who had Zika virus infection in pregnancy compared with those
who did not. In laboratory studies, Zika virus has been shown to cross the placenta
and replicate in human brain cells.
• For GBS, we assessed 36 studies that addressed questions about one or more causality
dimensions. In several countries in the Americas and the Pacific region, a temporal
association has been found, with symptoms of Zika virus infection preceding the
onset of GBS. In these countries, surveillance reports of cases of GBS followed the
pattern of reports of Zika-like illness. During a Zika virus outbreak in French Poly-
nesia in 2013–14, scientists estimated that around one in 4000 people with Zika virus
infection developed GBS. The odds of having had a recent Zika virus infection were
more than 30 times higher in patients with GBS than those without in a hospital-
based study in French Polynesia. Several other infections that can trigger GBS were
excluded.
What Do These Findings Mean?
• This systematic review found sufficient evidence to conclude that Zika virus is a cause of
congenital abnormalities and is a trigger of GBS.
• Systematic reviews of evidence about emerging public health threats need to be updated
frequently.
Introduction
An “explosive pandemic of Zika virus infection” [1] in 2015 caught the world by surprise. The
Pan American Health Organization (PAHO) and World Health Organization (WHO) pub-
lished an alert about a possible association with increases in reports of congenital abnormalities
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 3 / 27
and Guillain–Barre syndrome (GBS) on December 1, 2015 [2]. On February 1, 2016, WHO
declared a Public Health Emergency of International Concern [3]. Microcephaly at birth is a
clinical finding that can include a range of brain malformations resulting from a failure of neu-
rogenesis [4]. Infections acquired in pregnancy, including cytomegalovirus and rubella, are
established causes [4]. GBS is an immune-mediated ascending flaccid paralysis, which typically
occurs within a month of an infection, such as Campylobacter jejuni or cytomegalovirus [5]. As
of October 20, 2016, 67 countries have reported autochthonous transmission of the mosquito-
borne flavivirus Zika since 2015, and 27 of these countries have reported cases of congenital
brain abnormalities, GBS, or both [6]. The emergency committee recommended increased
research [3] to provide more rigorous scientific evidence of a causal relationship as a basis for
the global health response.
Unexplained clusters of rare but serious conditions require urgent assessment of causality,
balancing speed with systematic appraisal. Bradford Hill is widely credited for his proposed
framework for thinking about causality in epidemiology, which listed nine “viewpoints” from
which to study associations between exposure and disease (S1 Text, p2) [7]. Since then, the list
has been modified (S1 Text, p2; S1 Table) [8]. Bradford Hill emphasised that his viewpoints
were not rules but, taken together, the body of evidence should be used to decide whether
there is any other more likely explanation than cause and effect.
The level of certainty required before judging that Zika virus is a cause of microcephaly and
GBS is contentious [9]. Most assessments have been based on rapid but nonsystematic apprais-
als [10–12]. Based on rapid reviews, WHO has stated that there is “scientific consensus that
Zika virus is a cause of microcephaly and GBS” since March 31, 2016 [13]. On April 13, a nar-
rative review stated that there was “a causal relationship between prenatal Zika virus infection
and microcephaly” [11]. Evidence about the causal relationship between Zika virus and GBS
has not yet been assessed in detail. We previously described a causality framework for Zika
virus and plans for a systematic review (S1 Text, p3; S2 Table), with a preliminary overview of
21 studies, published up to March 4, 2016 [14]. The objectives of this study are to reassess the
evidence for causality and update the WHO position about links between Zika virus and (a)
congenital brain abnormalities, including microcephaly, in the foetuses and offspring of preg-
nant women and (b) GBS in any population, and to describe the process and outcomes of an
expert assessment of the evidence.
Methods
We describe three linked components: the causality framework, the systematic reviews, and
the expert panel assessment of the review findings. The WHO Zika Causality Working Group
convened the expert panel of 18 members with specialist knowledge in the fields of epidemiol-
ogy and public health, virology, infectious diseases, obstetrics, neonatology, and neurology
(membership of the expert panel is provided in the Acknowledgments).
Zika Causality Framework
In February 2016, we developed a causality framework for Zika virus by defining specific ques-
tions for each of ten dimensions, modified from Bradford Hill’s list (S2 Table): temporality;
biological plausibility; strength of association; exclusion of alternative explanations; cessation;
dose–response relationship; animal experimental evidence; analogy; specificity; and consis-
tency of findings. This review covered 35 questions about congenital brain abnormalities,
including microcephaly, and 26 questions about GBS. We also listed seven groups of cofactors
that might increase the risk of an outcome in the presence of Zika virus [15].
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 4 / 27
Systematic Review
Our protocol was registered on March 21, 2016 in the database PROSPERO (CRD42016036693)
[16]. We report the methods in full according to the Preferred Reporting Items for Systematic
Reviews and Meta-Analyses (PRISMA) [17] in S1 Text (p3-4).
To report our findings, we use the term item for an individual record, e.g., a case report.
Occasionally, the same individuals or data were reported in more than one publication (item).
To avoid double counting, we organised these items into groups. We chose a primary publica-
tion (the item with the most complete information) to represent the group, to which other
items were linked (S4 Table, S6 Table).
Eligibility. We included studies of any design and in any language that directly addressed
any research question in the causality framework (S1 Text, p3).
Information sources and search strategy. We searched multiple electronic databases and
websites (protocol [16] and S1 Text, p3) and included published peer-reviewed studies, ongo-
ing studies, and non-peer reviewed sources. For the dimension addressing analogous causes of
the outcomes and for cofactors, we did not conduct systematic searches.
We conducted our first search from the earliest date to April 11, 2016 and updated the
search on May 30 and July 29. We selected items and extracted data systematically on included
items up to May 30 and reported on nonsystematically identified studies up to July 29, 2016.
Study selection and data extraction. We screened titles, abstracts, and full texts by liberal
accelerated screening (S1 Text, p3) [18]. For data extraction, one reviewer extracted data and a
second reviewer checked the extracted data. We used case definitions and laboratory diagnos-
tic test interpretations as reported by study authors.
Synthesis of findings and assessment of methodological quality. We tabulated study-
level data and clinical information from case reports, case series, cross-sectional studies, case-
control studies, and cohort studies. We assessed methodological quality for these designs using
checklists [19]. Each reviewer recorded an overall judgement to indicate whether study find-
ings did or did not provide support for each causality dimension. We assigned a judgement of
sufficient evidence about a causality dimension if the consensus assessments were supportive
for at least half of the specific questions. We appraised the body of evidence according to the
Grading of Research Assessment Development and Evaluation (GRADE) tool, as suggested for
urgent health questions [20], but did not apply upgrading or downgrading because these con-
cepts could not be applied consistently across the range of study designs.
Expert Panel
In a series of web and telephone conferences between April 18 and May 23, 2016, we presented
our approach to the assessment of causality, the causality framework, and our synthesis of evi-
dence to the expert panel. We discussed these topics with the experts during the conferences
and through email discussions. We then drafted summary conclusions about the most likely
explanation for the reported clusters of cases of microcephaly and GBS. The expert panel
members reached consensus statements to update the WHO position (Fig 1).
Results
We found 1,091 unique items published from 1952 to May 30, 2016 (S1 Fig, S3 Table). Most
excluded items were reviews or editorials and commentaries (44%, n = 479). We included 106
items from 87 groups (Table 1), of which 83% were published in 2016. For both outcomes, the
majority of items were clinical, individual-level case reports, case series, or population-level
surveillance data.
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 5 / 27
Fig 1. Timeline of Zika causality review, February 1 to August 2016. A Public Health Emergency of International Concern was announced on February 1,
2016 in response to clusters of microcephaly, GBS, and other neurological disorders.
doi:10.1371/journal.pmed.1002203.g001
Table 1. Summary of included items according to outcome, study design, and causality dimension.
Congenital abnormalities GBS
N % N %
Type of study
Case report 9 12.5 9 25
Case series 22 30.6 5 13.9
Case-control study 0 0 1 2.8
Cohort study 1 1.4 0 0
Cross-sectional study 2a 2.8 0 0
Ecological study/outbreak report 5 6.9 19 52.8
Modelling study 2 2.8 0 0
Animal experiment 18 25 0 0
In vitro experiment 10 13.9 0 0
Sequence analysis and phylogenetics 3 4.2 2 5.6
Total items 72 100 36 100
Causality dimensionb
Temporality 21 36.2 26 83.9
Biological plausibility 25 43.1 4 12.9
Strength of association 3 5.2 2 6.5
Alternative explanation 18 31 6 19.4
Cessation 2 3.4 6 19.4
Dose–response relationship 0 0 0 0
Experiment 20 34.5 0 0
Analogy NA NA NA NA
Specificity 0 0 0 0
Consistency NA NA NA NA
Total groupsc 58 31
a One cross-sectional study studied human participants and one studied monkeys.b A group of items could contribute to more than one causality dimension, so totals do not sum to 100%.c Two items contribute to both topics.
Abbreviations: NA, not applicable; evidence about analogous conditions was not searched systematically;
the dimension of consistency used information in items included for all other causality dimensions.
doi:10.1371/journal.pmed.1002203.t001
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 6 / 27
Congenital Brain Abnormalities
A total of 72 items belonging to 58 groups addressed questions related to congenital brain
abnormalities up to May 30, 2016 [13, 21–93]. Table 2 summarises the characteristics of 278
mother–infant pairs described in included studies.
Table 3 summarises the assessment for each causality dimension, S4 Table provides an
extended description of study findings, and S5 Table summarises the quality of the body of
evidence.
Temporality. Thirty-five items [30–37, 39–43, 45–47, 49, 51, 52, 57, 60, 65, 67, 68, 73–76,
78, 80, 85, 86, 88–90] in 21 groups addressed temporality (S4 Table). Overall, 67.9% (180/265)
of women reported symptoms of Zika virus infection during pregnancy (Table 2). Confirmed
infection preceded a diagnosis of microcephaly in a small proportion of cases because many
reports were published before laboratory confirmation testing was available. Of the 36 mothers
with laboratory-confirmed Zika virus infection, 19 (52.8%) were diagnosed before the detec-
tion of foetal malformations or miscarriage [40, 42, 45, 52, 67]. Two detailed case reports show
timelines of recent infection followed by neuroimaging evidence of brain abnormalities and
subsequent birth with microcephaly [42] or foetal infection [52]. The most likely time point of
exposure was the first or the early second trimester, based on individual case reports and three
modelling studies [47, 49, 60]. At the population level, epidemic curves of reported Zika virus
illness increased in parallel with reported cases of microcephaly, with a lapse of 30 to 34 wk in
two states of Brazil (Pernambuco and Bahia) (S2 Text) [57, 60].
Biological plausibility. Twenty-eight items [29, 30, 34, 36–38, 40, 41, 44, 51, 52, 54–58,
61, 63, 64, 67, 69, 72, 75, 77, 79, 81, 85, 91–93] in 25 groups addressed biological plausibility
(S4 Table). These studies suggest a teratogenic effect of Zika virus on the developing brain.
Detailed investigations about a woman with Zika virus infection whose pregnancy was termi-
nated found that isolated viral particles from the foetal brain, but not other tissues, were capa-
ble of replication in cell culture [52]. Zika virus RNA was also found in foetal brain tissue in
three other studies [34, 36, 38]. Zika virus from both the African and the Brazilian (Asian) line-
ages replicates in different types of neural progenitor cells (NPCs) [44, 69, 91]. The phosphati-
dylserine-sensing receptor tyrosine kinase AXL is a potential entry point into human cells and
is expressed in developing human cerebral cortex tissue [29, 64]. In vitro studies using NPCs
and cerebral organoids show that Zika virus replicates in neural tissue and can disturb the cell
cycle and lead to apoptosis [44, 69, 77, 81, 91].
Strength of association. We reviewed seven items [42, 46, 47, 49, 60, 78, 86] in three
groups up to May 30, 2016 (S4 Table). Two studies suggest that the association between Zika
virus infection in pregnancy and congenital brain abnormalities is likely to be very strong [42,
47]. In Rio de Janeiro, investigators compared 72 women with positive reverse transcriptase
PCR (RT-PCR) results for Zika virus with 16 women with other causes of rash [42]. Follow-up
was more intensive in women with Zika virus infection than those without. Of 42 Zika-
infected women with one or more ultrasound scans, 12 (29%) had abnormal scans. All 16
women without Zika virus infection were reported to have had one normal routine scan, but
no follow-up data were reported. The preliminary description of the data suggests a marked
increase in the risk of congenital abnormalities. In French Polynesia, investigators recon-
structed a hypothetical cohort of pregnant women from different sources of data, including
eight retrospectively identified cases of microcephaly [47]. They estimated that the risk of
microcephaly would be 53.4 times (95% confidence interval 6.5–1,061.2) higher in women
with Zika virus infection than in uninfected women if exposure had occurred in the first tri-
mester. Methods and assumptions were clearly described, but the estimate was imprecise and
was obtained from indirect data sources. One case-control study in Pernambuco, Brazil, was
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 7 / 27
Table 2. Geographic, clinical, and microbiological characteristics of mother–infant pairs.
Characteristic References No. with
characteristicaNo. evaluated in the
articlea%b
Total with congenital abnormalities or adverse
pregnancy outcomes
278 278 100
Country of infection
Brazil [32, 35, 36] 242 278 87.1
Cabo Verde [13, 66] 2 278 0.7
Colombia [45] 2 278 0.7
French Polynesia [51] 19 278 6.8
Martinique [67] 1 278 0.4
Panama [55–57] 4 278 1.4
Travellers returning from the Americas [34, 40, 52, 85] 8 278 2.9
Pregnancy outcome
Miscarriage [36, 40, 42, 79] 7 278 2.5
Intrauterine death or stillbirth [38, 42] 3 278 1.1
Termination of pregnancy [34, 40, 42, 51, 52] 15 278 5.4
Neonatal death [36, 51, 55–57, 61, 79] 9 278 3.2
Alive, still in utero [13, 32, 35, 37, 39, 40, 42, 51, 55–57, 66–
68, 80, 85, 88–90]
8 278 2.9
Live birth [13, 32, 35, 37, 39, 40, 42, 51, 55–57, 66–
68, 80, 85, 88–90]
236 278 84.9
Time point of presumed exposure (symptoms)
1st trimester [32, 34–37, 39, 40, 42, 51, 52, 79, 85, 88–
90]
81 117 69.2
2nd trimester [32, 35, 37, 42, 80, 88] 28 117 23.9
3rd trimester [35, 42, 79, 88] 8 117 6.8
Exposure assessment in the mother
Zika virus (ZIKV)-related clinical symptoms [32, 34–40, 42, 45, 51, 52, 55–57, 68, 79,
80, 85, 88–90]
180 265 67.9
ZIKV positive in any test (serology/PCR/IHC) [13, 34, 37, 38, 40, 42, 45, 51, 52, 55–57,
66, 67, 80, 85, 90]
36 41 87.8
ZIKV positive in any test before the outcome [13, 34, 37, 38, 40, 42, 45, 51, 52, 55–57,
66, 67, 80, 85, 90]
19 36 52.8
ZIKV IgM positive (serum) [13, 34, 37, 52, 66, 85, 90] 3 7 42.9
ZIKV IgG positive (serum) [13, 34, 52, 66] 3 3 100.0
ZIKV PRNT positive (serum) [34, 52, 85, 90] 4 4 100.0
ZIKV RT-PCR positive (serum) [37, 45, 52, 80, 90] 3 7 42.9
ZIKV RT-PCR positive (urine) [37, 52, 55–57, 90] 1 5 20.0
ZIKV RT-PCR positive (amniotic fluid) [37, 38, 51, 52, 67] 9 12 75.0
DENV IgG positive [34, 37, 39, 42, 45, 51, 52, 80, 90] 17 28 60.7
Exposure assessment in the foetus/newborn
ZIKV positive in any test (serology/PCR/IHC) [13, 34, 36, 38, 40, 52, 61, 66, 68, 79, 80,
85, 88, 90]
74 75 97.4
ZIKV IgM positive (serum) [68, 80, 85, 90] 30 34 88.2
ZIKV IgG positive (serum) [13, 34, 66, 80, 90] 4 4 100.0
ZIKV PRNT positive (serum) [85, 90] 2 2 100.0
ZIKV RT-PCR positive (serum) [61, 67, 68, 90] 2 34 5.9
ZIKV RT-PCR positive (brain tissue) [34, 36, 38, 52, 79] 6 6 100.0
ZIKV RT-PCR positive (other tissue) [34, 36, 38, 40, 52, 55–57, 61, 80] 6 11 54.5
ZIKV RT-PCR positive (placenta/product of
conception)
[36, 40, 52, 79, 85] 7 8 87.5
(Continued )
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 8 / 27
ongoing at the time of the first searches. The Microcephaly Epidemiology Research Group
enrolled 32 cases and 62 controls and found a crude odds ratio of 55.0, 95% CI 8.66–1) between
neonatal microcephaly and laboratory-confirmed Zika virus infection in pregnancy [94].
At population level, state-level data in Brazil showed a positive correlation between case
reports of Zika-like illness and cases of microcephaly [49]. These data also show a higher prev-
alence of microcephaly in 15 states that had reported cases (2.8 per 10,000 live births) than in
four states with no reported cases (0.6 per 10,000 live births) [46] (prevalence ratio 4.7, 95% CI
1.9–13.3).
Exclusion of alternative explanations. Twenty-eight items [30–39, 41–43, 45, 51, 52, 65,
68, 73–76, 79, 80, 85, 88–90] in 18 groups addressed alternative explanations (S4 Table). No
alternative single infectious cause could have resulted in large clusters of cases of microcephaly
in different places. Acute dengue virus infection was excluded in 12 studies. Four studies
excluded maternal exposure to alcohol or medication, or genetic causes of congenital abnor-
malities [34, 35, 37, 51]. No study excluded exposure to environmental toxins or heavy metals.
Cessation. Six items [46, 49, 57, 60, 78, 86] in two groups addressed this dimension (S4
Table). Surveillance reports of Zika-like illness in northeastern Brazil in 2015 declined [57, 60]
either due to seasonality of the vector or population immunity. Reports of microcephaly
declined with a similar pattern in Bahia state [60]. In Pernambuco state, a similar pattern was
observed but a dengue epidemic occurred simultaneously, so the decline in microcephaly
cases might not be attributable to the Zika virus outbreak alone (S2 Text) [57]. We did not find
any data on trends in microcephaly cases in countries other than Brazil.
Dose–response relationship. We did not find any relevant studies.
Experiments in animals. We reviewed 20 items [21–28, 48, 50, 53, 59, 62, 69–71, 82–84,
87] (S4 Table). Studies in the 1950s–1970s show that experimental inoculation of Zika virus
Table 2. (Continued)
Characteristic References No. with
characteristicaNo. evaluated in the
articlea%b
Total with congenital abnormalities or adverse
pregnancy outcomes
278 278 100
ZIKV RT-PCR positive (CSF) [38, 85, 88] 26 26 100.0
ZIKV IHC positive (brain) [34, 36, 52, 79] 4 5 80.0
ZIKV IHC positive (other tissue) [34, 36, 40, 79] 2 7 28.6
ZIKV IHC positive (placenta/product of conception) [40, 79, 85] 3 4 75.0
DENV IgG positive [68, 90] 1 34 2.9
Outcome assessment
Clinical microcephaly [13, 32, 34–40, 42, 51, 55–57, 61, 66–68,
79, 80, 85, 88–90]
244 267 91.4
Imaging confirmed brain abnormalities [32, 34, 37–40, 42, 45, 51, 52, 55–57, 68,
80, 85, 88–90]
205 213 96.2
Intrauterine growth restriction [34, 38, 39, 42, 51, 85] 10 35 28.6
Ocular disorders [35, 37, 40, 42, 51, 68, 80, 85, 88, 89] 49 116 42.2
Auditory disorders [51, 68] 3 24 12.5
Abnormal amniotic fluid [42, 51, 61, 80] 6 33 18.2
a The denominator for each characteristic is the number of cases for which data were available.b Column percentages shown for country of infection, pregnancy outcome, and time point of exposure; row percentages for all other variables.
Abbreviations: CSF, cerebrospinal fluid; DENV dengue virus; IHC, immunohistochemistry; Ig, immunoglobulin; PRNT, plaque reduction neutralisation test;
RT-PCR, reverse transcriptase PCR; ZIKV, Zika virus.
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Table 3. Summary of reviewers’ assessments of evidence about Zika virus infection and congenital
abnormalities, by causality dimension.
Causality dimensiona Number of items
and groupsbEvidence summaryc
Temporality 35 items in 21
groups
Reviewer assessments found sufficient evidence for all
three questions of an appropriate temporal relationship
between Zika virus (ZIKV) infection and the occurrence of
congenital abnormalities, including microcephaly. The
period of exposure to ZIKV was most likely to be in the
first or early second trimester of pregnancy.
Biological plausibility 28 items in 25
groups
Reviewer assessments found sufficient evidence for six
of seven questions that address biologically plausible
mechanisms by which ZIKV could cause congenital
abnormalities.
Strength of association 7 items in 3 groups Reviewer assessments found sufficient evidence of a
strong association between ZIKV infection and congenital
abnormalities for two of two questions. At the population
level, there is strong evidence of an association. At the
individual level, the effect size was extremely high,
although imprecise, in one study and is likely to be high in
the other study when follow-up is complete. A newly
published case-control study from Brazil shows an effect
size similar to that of the retrospective study from French
Polynesia.
Exclusion of alternative
explanations
28 items in 18
groups
Reviewer assessments found sufficient evidence at the
individual level that alternative explanations have been
excluded for three of seven questions; no other single
explanation could have accounted for clusters of
congenital abnormalities. The evidence about other
exposures could not be assessed because of an absence
of relevant studies.
Cessation 6 items in 2 groups Reviewer assessments found sufficient evidence for one
of three questions. In two states of Brazil and in French
Polynesia, cases of congenital abnormalities decreased
after ZIKV transmission ceased. Evidence for the other
questions could not be assessed because no relevant
studies were identified.
Dose–response
relationship
0 items This dimension could not be assessed because of an
absence of relevant studies.
Animal experiments 20 items in 20
groups
Reviewers assessments found evidence from animal
experimental studies for all four questions that supports a
causal link between ZIKV and congenital abnormalities.
Inoculation with ZIKV of pregnant rhesus macaques and
mice can result in foetal abnormalities, viraemia, and
brain abnormalities. Experiments to induce viral
replication after inoculation of ZIKV intracerebrally and at
other sites in a variety of animal models have produced
mixed results.
Analogy Not reported Selected studies reviewed. There are analogies with the
well-described group of TORCH infections. Microcephaly
has been described following the flavivirus West Nile virus
(WNV) infection in pregnancy but not DENV. Evidence
was not reviewed systematically.
Specificity 0 items We did not find any studies that identified congenital
abnormalities that were found following Zika virus
infection in pregnancy but not in other congenital
infections. The studies included described a wide range of
abnormalities on clinical and neuroimaging examinations.
Many of the abnormalities described are also found in
other congenital infections, but with a different pattern.
(Continued )
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resulted in illness, cerebral lesions, and viral replication in the brain in some but not all species
tested [21–25, 27, 28]. Some effects might have been enhanced by the numerous serial passag-
ing and subsequent viral adaptation of the original Ugandan Zika strain MR766 and the choice
of genetically susceptible animal models. More recent animal studies have shown evidence of
neurotropism in immunocompromised mice and in foetal or infant (suckling) immunocom-
petent mice [48, 71, 82] but not in adult immunocompetent mice [50, 53]. Real-time reports
are documenting studies of Macaque monkeys experimentally infected with a Brazilian strain
and a French Polynesian strain of Zika virus (both Asian lineage) during pregnancy [70]. High
and persisting viraemia was observed in one animal. Foetal autopsy revealed viral RNA in
some tissues, but the brain tissue was negative for Zika virus and showed no histopathological
lesions or clinical microcephaly.
Analogy. Clinical observations linking clusters of babies born with microcephaly and an
earlier outbreak of Zika virus infection in Brazil are analogous to the discovery in 1941 of con-
genital rubella syndrome [95]. Cytomegalovirus and toxoplasmosis in pregnancy can both
cause microcephaly, intracranial calcification, and ocular and auditory defects [96] (cited in
[43]). Two cases of microcephaly were reported amongst 72 women infected with the neuro-
tropic flavivirus West Nile virus infection in pregnancy [97]. A review of 30 studies of dengue
virus infection in pregnancy found evidence of vertical transmission but did not mention
microcephaly or other congenital brain abnormalities as possible complications [98].
Specificity of association. We did not find any studies that described neuroimaging or
clinical features found only in association with Zika virus infection.
Consistency. Findings that support Zika virus infection as a cause of congenital brain
abnormalities have come from different kinds of epidemiological studies and laboratory stud-
ies in both humans and animals (S4 Table). Case reports of pregnancies affected by Zika virus
Table 3. (Continued)
Causality dimensiona Number of items
and groupsbEvidence summaryc
Consistency Not reported For three of four questions, the evidence assessed was
consistent. By geographical region, maternal exposure to
ZIKV has been associated with the occurrence of
congenital abnormalities in three regions. By study
design, the association between ZIKV infection and
congenital abnormalities has been found in studies at
both individual and population levels and with both
retrospective and prospective designs. By population
group, ZIKV infection has been linked to congenital
abnormalities in both women resident in affected
countries and in women from nonaffected countries
whose only possible exposure to ZIKV was having
travelled in early pregnancy to an affected country. The
evidence according to ZIKV lineage is inconsistent
because an association between ZIKV and congenital
abnormalities has only been reported from countries with
ZIKV of the Asian lineage since 2013.
a Questions for each causality dimension are in S2 Table.b Number of items not reported for Analogy because evidence was not searched for systematically and for
Consistency because the evidence about this dimension draws on items that contribute to all other
dimensions.c The complete evidence table is in S4 Table.
Abbreviations: DENV, dengue virus; TORCH, Toxoplasmosis, Rubella, Cytomegalovirus, Herpes simplex
virus; WNV, West Nile virus; ZIKV, Zika virus.
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have come from the Americas, the Pacific region (Table 2), and West Africa [13, 66]. The prev-
alence of microcephaly has not been higher than expected in all countries with Zika virus
transmission, however. Congenital brain abnormalities or the presence of Zika virus in prod-
ucts of conception has also been found in pregnant travellers returning from Zika-affected
countries [34, 40, 52], showing consistency across populations. There have been no reports of
congenital brain abnormalities from countries affected by the African lineage [99]. One in
vitro study found that Brazilian (Asian lineage) and African Zika strains both replicated in
murine and human cell cultures and organoids [69, 77].
Summary of quality of evidence. The body of evidence includes a wide range of study
designs and populations in both humans and animals (S5 Table). Much of the evidence in
humans comes from uncontrolled or ecological study designs that have inherent biases for
ascertaining causal associations. Of two studies that quantified the strength of association,
effect sizes were very large but also imprecise [47, 94]. One of three comparative studies was at
low risk of bias [94]. Evidence from animal studies is, by its nature, indirect. We could not for-
mally assess publication bias; our search strategy was wide, but we found very few studies with
findings that were not consistent with causality.
Guillain–Barre Syndrome
We found 36 items belonging to 31 groups that addressed questions related to GBS [54–57, 67,
78, 100–122]. We summarise the findings according to clinical characteristics of 118 individu-
als diagnosed with GBS in Table 4.
Table 5 summarises the reviewers’ assessments by causality dimension, S6 Table provides
an extended description of study findings, and S7 Table summarises the quality of the body of
evidence.
Temporality. We included 31 items [55–57, 67, 78, 100–112, 115–117, 120–122] in 26
groups (S6 Table). At the individual level, symptoms of Zika virus infection were reported
before the onset of GBS symptoms in cases in French Polynesia, Brazil, El Salvador, Panama,
Puerto Rico, and Venezuela, and in returning travellers from Haiti, Suriname, and Central
America. All patients with GBS had laboratory-confirmed Zika virus infection except for 42 of
44 in Brazil and all those in El Salvador. The intervals between Zika virus infection and neuro-
logical symptoms delays of 3 to 12 d [108, 111, 112] are consistent with a postinfectious auto-
immune mechanism [5]. In one ecological study in Bahia, Brazil, a lag of 5 to 9 wk between the
epidemic peaks of cases with acute exanthematous illness and GBS was attributed to data col-
lection issues [78].
At the population level, 11 countries in Latin America (Brazil, Colombia, El Salvador, French
Guiana, Honduras, Venezuela, Suriname) and the Caribbean (Dominican Republic, Jamaica,
Martinique) and French Polynesia have reported an increase in GBS cases during outbreaks of
Zika virus infection [56, 57, 67, 104, 107, 109]. Surveillance reports show sporadic GBS cases in
association with Zika-like illness in four countries but without an increase above background
level (Guadeloupe, Haiti, Panama, Puerto Rico). One study reported on surveillance data about
acute flaccid paralysis in children in 20 South Pacific islands. The number of expected cases of
acute flaccid paralysis was<1 per year in these small countries, and an increase during periods
of Zika virus transmission was only observed in the Solomon Islands [115].
Biological plausibility. We reviewed six items [54, 102, 111, 112, 114, 116] in four groups
(S6 Table). Anti-ganglioside antibodies, whose presence supports the clinical diagnosis of
GBS, were found in the serum of a third of patients in a case-control study in French Polynesia
[112] and in one patient from Venezuela [111]. The case-control study and two in silico studies
also provide some evidence for molecular mimicry of Zika virus epitopes and host antigens
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[112]. Studies of predicted epitopes and human antigens suggested peptide sharing between
Zika virus and human proteins [54, 114]. Several experimental studies with human neural
stem cells and mouse models have shown some evidence for neurotropism of Zika virus (S4
Table, biological plausibility).
Strength of association. We reviewed seven items [101–104, 112, 116, 122] in two groups
identified up to May 30, 2016. One published case-control study enrolled 42 cases of GBS dur-
ing the Zika outbreak in French Polynesia and compared them with 98 patients hospitalised
with nonfebrile illness (S6 Table) [112]. Several alternative causes of GBS were excluded. Evi-
dence of Zika virus infection was much more common in GBS cases than controls (odds ratios
59.7, 95% CI 10.4–+1 defined as IgM or IgG positivity and 34.1, 95% CI 5.8–+1 defined as
presence of neutralising antibodies). Cases and controls were matched, but there was no addi-
tional adjustment for confounding. In Brazil, surveillance data showed a 19% increase in
reports of GBS cases in 2015 compared with 2014 [101]. Information received after May 30
found a second case-control investigation conducted in Brazil that enrolled controls from the
community and is ongoing; preliminary results suggest a similar, strong effect (Sejvar J., per-
sonal communication).
Table 4. Geographic, clinical, and microbiological characteristics of people with GBS.
References No. with characteristic No. evaluated %
Total N of cases with GBS 118 118 100
Country of infection
Brazila [105, 117, 118] 44 118 37.3
El Salvadora [108] 22 118 18.6
French Polynesia [112] 42 118 35.6
Haiti [119] 1 118 0.8
Martinique [113] 2 118 1.7
Panama [106, 108] 2 118 1.7
Puerto Rico [110] 1 118 0.8
Travellers returning from the Americas [108, 120] 3 118 2.5
Venezuela [111] 1 118 0.8
Exposure assessment
Zika virus (ZIKV) symptomatic cases [105, 106, 108, 110–112, 117, 118, 120] 84 113 74.3
ZIKV positive in any test (serology/RT-PCR) [106, 108, 110–113, 117–120] 54 54 100.0
ZIKV IgM positive (serum) [110, 112, 119] 41 44 93.2
ZIKV IgG positive (serum) [112] 29 42 69.0
ZIKV PRNT positive (serum) [112, 119] 43 43 100.0
ZIKV RT-PCR positive (serum) [106, 108, 110, 112, 113, 118, 120] 4 50 8.0
ZIKV RT-PCR positive (urine) [106, 108, 110, 113, 120] 6 7 85.7
ZIKV RT-PCR positive (saliva) 0 0 -
ZIKV RT-PCR positive (CSF) [106, 108, 113, 118] 2 4 50.0
ZIKV culture positive (serum) 0 0 -
ZIKV culture positive (CSF) 0 0
DENV IgG positive [110, 112, 113, 120] 43 45 95.6
Interval between ZIKV and GBS symptoms, days Median 10, range 3–12 [106, 108, 110, 111, 117, 120]
French Polynesia: Median 6 (IQR 4–10) [112]El
Salvador: 7–15 [108]
a Only one patient with GBS in Brazil and none in El Salvador had laboratory confirmation of Zika virus infection.
Abbreviations: CSF, cerebrospinal fluid; DENV dengue virus; IQR, interquartile range; Ig, immunoglobulin; PRNT, plaque reduction neutralisation test;
RT-PCR, reverse transcriptase PCR; ZIKV, Zika virus.
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Alternative explanations. We included ten items [102, 110, 112, 113, 116–121] in seven
groups (S6 Table). In several studies, other infections that can trigger GBS were excluded, such
as C. jejuni, Mycoplasma pneumoniae, HIV, Epstein–Barr virus, and herpes simplex virus. No
single infectious trigger would have resulted in GBS outbreaks in multiple locations.
Cessation. Eight items [56, 57, 78, 103, 104, 109, 122] in six groups addressed cessation
(S6 Table). In surveillance reports from six countries (Brazil, Colombia, El Salvador, French
Table 5. Summary of reviewers’ assessments of evidence about Zika virus infection and GBS, by causality dimension.
Causality dimensiona Number of items and
groupsbEvidence summaryc
1. Temporality 31 studies in 26
groups
Reviewer assessments found sufficient evidence for all three questions of an appropriate
temporal relationship between ZIKV infection and GBS. The time interval between ZIKV
symptoms and onset of neurological symptoms was compatible with that of other accepted
triggers of GBS.
2. Biological plausibility 6 items in 4 groups Reviewer assessments found sufficient evidence for two of three questions about biologically
plausible mechanisms by which ZIKV could trigger the immune-mediated pathology of GBS.
There is evidence that supports a role for molecular mimicry, a proposed mechanism of
autoimmunity, which has been reported in C. jejuni-associated GBS. Direct neurotropic effects of
ZIKV might also occur.
3. Strength of association 7 items in 2 groups The reviewers assessed evidence from the ZIKV outbreak in French Polynesia as showing a
strong association between ZIKV and GBS at both the individual and population levels.
Surveillance reports from Brazil also support an association at the population level. Preliminary
results from a case-control study in Brazil suggest a similar, strong effect.
4. Exclusion of alternative
explanations
10 items in 7 groups Reviewer assessments found sufficient evidence at the individual level that other infectious
triggers of GBS have been excluded; no other single infection could have accounted for clusters
of GBS. The evidence about other exposures could not be assessed because of an absence of
relevant studies.
5. Cessation 8 items in 6 groups Reviewer assessments found sufficient evidence for one of three questions. In one state in Brazil,
four other countries in the Americas, and in French Polynesia, reports of GBS decreased after
ZIKV transmission ceased. Evidence for the other questions could not be assessed because no
relevant studies were identified.
6. Dose–response
relationship
0 items No relevant studies identified.
7. Animal experiments 0 items No relevant studies of animal models of immune-mediated neuropathology identified. Evidence
about neurotropism of ZIKV summarised in S4 Table.
8. Analogy Not reported Evidence was not reviewed systematically; selected studies reviewed for two of three questions.
Analogous mosquito-borne neurotropic flavivirus infections have been reported in association
with GBS (WNV; DENV; JEV). WNV and JEV have also been reported to be associated with
direct neurotropic effects and poliomyelitis-like acute flaccid paralysis. The time lag between ZIKV
symptoms and GBS symptoms is analogous to intervals reported for other infectious triggers of
GBS. There is some evidence that, as for C. jejuni-associated GBS, molecular mimicry could be
involved.
9. Specificity 0 items No relevant studies identified.
10. Consistency Not reported For three of four questions, there was sufficient evidence of consistency. By geographical region,
ZIKV transmission has been associated with the occurrence of GBS in two of three regions where
ZIKV has circulated since 2007. By study design, the association between ZIKV infection and
GBS has been found in studies at both individual and population levels. By population group,
ZIKV infection has been linked to GBS in both residents of an affected country and travellers from
nonaffected countries whose only possible exposure to ZIKV was having travelled to an affected
country. The evidence according to ZIKV lineage is unclear because an association between
ZIKV and GBS has only been reported from countries with ZIKV of the Asian lineage since 2013.
a Questions for each causality dimension are in S2 Table.b Number of items not reported for dimension 8 (Analogy), because evidence was not searched for systematically, and for Consistency, because the
evidence about this dimension draws on items that contribute to all other dimensions.c The complete evidence table is in S6 Table
Abbreviations: DENV, dengue virus; GBS, Guillain–Barre syndrome; JEV, Japanese encephalitis virus; WNV, West Nile virus; ZIKV, Zika virus.
doi:10.1371/journal.pmed.1002203.t005
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Polynesia, Honduras, and Suriname), the incidence of GBS declined as reports of Zika virus
infection fell.
Dose–response relationship, experiments in animals, and specificity. We did not find
any relevant studies.
Analogy. Clusters of cases of GBS have been reported in association with outbreaks of C.
jejuni gastroenteritis [123]. The incidence of GBS estimated from studies in French Polynesia
of 0.24 per 1,000 Zika virus infections [112] is at the lower end of estimates for C. jejuni (0.3
[124] and 1.17 [125] per 1,000). The reported latency between gastrointestinal symptoms and
onset of paralysis of approximately 9 d (range 1–23 d) [124, 126, 127] is similar to Zika virus-
associated cases. Other mosquito-borne neurotropic flaviviruses have been reported as possi-
ble triggers of GBS in case reports and case series: dengue virus [128], West Nile virus [129],
Japanese B encephalitis virus [130, 131], or yellow fever 17D vaccination [132]. An acute polio-
myelitis-like flaccid paralysis, resulting from direct neural infection, presumably of anterior
horn cells, has also been reported as a clinical consequence of these viruses [129, 133, 134].
Putative biological mechanisms include up-regulation of major histocompatibility class I and
II molecules of peripheral nerve cells and subsequent immune-mediated cell destruction [135],
auto-antibodies directed against heat shock proteins [136], galactocerebrosides [137], or mye-
lin basic protein (MBP), and proliferation of MBP-specific T-cells [138].
Consistency. The link between Zika virus and GBS has been made in studies of different
designs at individual and population levels (S6 Table). Clusters of GBS have been seen in mul-
tiple countries during epidemics of Zika virus but have not been reported in all those in which
Zika virus outbreaks have occurred. Outbreaks of GBS in which gene sequencing has been
done were associated with Zika virus of the Asian lineage.
Summary of quality of evidence. The body of evidence includes a wide range of study
designs and populations in humans (S7 Table). A majority of the evidence reviewed was from
uncontrolled or ecological study designs that have inherent biases for ascertaining causal asso-
ciations. The only study that examined the strength of association found a very large but
imprecise estimate of the effect size but did not have serious risks of bias [112]. There was no
evidence of indirectness. We could not formally assess publication bias, but we had a broad
search strategy, and we did find evidence that outbreaks of GBS have not been seen in all coun-
tries with Zika virus transmission.
Cofactors that might act in the presence of Zika virus. We prespecified seven categories
of cofactors (S2 Table). The most widely discussed was past dengue virus infection [112]. A
mechanism known as antibody-dependent enhancement might be involved, when IgG anti-
bodies against viral envelope proteins resulting from a prior infection bind to virus particles of
a subsequent infection, leading to enhanced replication and potentially more severe illness
[139]. Evidence from in vitro experiments suggests cross-reactivity between dengue and Zika
virus antibody responses and antibody-dependent enhancement of Zika virus by dengue anti-
bodies [139, 140]. In several of the studies that we reviewed, evidence of past dengue virus
infection was reported (S1 Text, p4-5). We did not systematically review evidence for other
cofactors but report additional narrative findings in S1 Text (p4-5).
WHO Expert Panel Conclusions
Based on the evidence identified up to July 29, 2016, the expert panel concluded that
• the most likely explanation of available evidence from outbreaks of Zika virus infection and
clusters of microcephaly is that Zika virus infection during pregnancy is a cause of congenital
brain abnormalities including microcephaly and
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• the most likely explanation of available evidence from outbreaks of Zika virus infection and
GBS is that Zika virus infection is a trigger of GBS [141].
The expert panel recognises that Zika virus alone may not be sufficient to cause either con-
genital brain abnormalities or GBS. The panel does not know whether these effects depend on
as yet uncharacterised cofactors being present, nor does it know whether dengue virus plays a
part, as this is carried by the same species of mosquito and has circulated in many countries
during the same period.
Discussion
Up to May 30, 2016, we found evidence that supported a causal association between Zika virus
infection and congenital brain abnormalities, including microcephaly, with at least one study
addressing one or more specific questions for eight of ten causality dimensions and between
Zika virus infection and GBS, with at least one study about one or more specific questions in
seven of ten dimensions. There are methodological weaknesses, inconsistencies, and gaps in
the body of evidence for both sets of conditions. Studies found after the cut-off for our first
searches did not change our conclusions but strengthened the evidence about biological plau-
sibility, strength of association, and exclusion of alternative explanations.
Interpretation of the Review Findings
The expert panel’s conclusions support causal links between Zika virus and congenital brain
abnormalities and GBS and address Bradford Hill’s question, “. . .is there any other answer
equally, or more, likely than cause and effect?” [7]. The conclusions consider both the epidemi-
ological context of unexpected clusters of different types of neurological conditions in coun-
tries that have experienced their first outbreaks of Zika virus infection and the strengths and
weaknesses of a systematically reviewed body of evidence about ten dimensions of causality
(S4 Table, S5 Table, S6 Table and S7 Table). Empirical observations cannot “prove” causality,
however [7, 142], and discussions have been intense [9]. A cause can be identified without
understanding all the necessary component causes or the complete causal mechanisms
involved [142, 143]. In the case of GBS, the infections that precede it are often referred to as
“triggers” of the immune-mediated causal pathways involved in pathogenesis.
The body of evidence about Zika virus and congenital abnormalities (72 items included)
has grown more quickly than that for GBS (36 items). Research efforts might have concen-
trated on congenital abnormalities because clusters of affected infants were so unusual, espe-
cially in Brazil, where rubella has been eradicated. In contrast, GBS is an established
postinfectious neurological disorder, and some commentators have already assumed Zika
virus as another cause [12]. Whilst only one case-control study from French Polynesia has
been published so far [112], clusters of GBS during outbreaks of Zika virus infection have been
reported from several other countries [144], and case-control studies are ongoing in Brazil,
Colombia, Mexico, and Argentina.
Comparative studies from French Polynesia suggest that the risk of both microcephaly or of
GBS is at least 30 times higher in people who had Zika virus infection compared to those who
did not [47, 94, 112], although confidence intervals are wide. The true effect size might be
weaker because the earliest studies investigating causality are often overestimates [145]. Even if
the methods of forthcoming studies in Brazil [42] and elsewhere reduce confounding and bias,
the increase in the risk of disease amongst those with Zika virus infection is likely to remain
substantially raised. Inconsistencies in the evidence still need investigation, however. Disease
clusters were not seen in Africa [146], but congenital abnormalities and GBS are rare
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complications that might not be detected in countries with small populations or poor surveil-
lance systems. In the case of microcephaly, terminations of potentially affected pregnancies
might have resulted in underascertainment [147].
Current evidence does not show which specific environmental and host factors interact
with Zika virus. A cofactor that increases the risk of neurological damage could help to explain
why surveillance reports show clusters of microcephaly or GBS in some geographical areas but
not others. Dengue virus has been suggested as a possible cofactor or another component
cause [143]. One major limitation to interpretation of data about causality and cofactors is the
lack of accurate and accessible diagnostic tools, owing to the short duration of viraemia, cross-
reactivity with other flaviviruses, and lack of standardisation [148].
Strengths and Limitations
The strengths of our study are that we appraised evidence of causality systematically but rap-
idly and transparently within a structured framework. We searched both published and
unpublished sources. The systematic review process could not eliminate publication bias but
reduced the risk that only positive reports in favour of causation would be evaluated. There
were limitations to the process, too. Our search strategy did not cover the literature about anal-
ogous conditions or cofactors systematically. We did not select studies or extract data in dupli-
cate, but additional reviewers checked the extracted data independently. The included studies
used a variety of case definitions for microcephaly and GBS, and we could not standardise
these, so misclassification is possible, but this limitation did not change the overall conclusions.
Our rapid assessment of quality was not quantitative; we did not find a tool that covered all
review questions and study designs appropriately and were not able to standardise the GRADE
tool across study designs in the time available [20].
Implications for Policy and Research
The conclusions of the expert panel facilitate the promotion of stronger public health mea-
sures and research to tackle Zika virus and its effects. The evidence gaps that we identified
provide researchers with research questions, and WHO has published a Zika virus research
agenda [149]. Better diagnostic tests will allow more accurate assessment of Zika virus in
tissues and of population-level immunity. Research about Zika virus and acute flaccid paral-
ysis is needed to define the clinical and electrophysiological pattern, mechanisms of causal-
ity, and to distinguish between the roles of autoimmunity and direct effects on anterior
horn cells or neurons. Basic research will also further the development of vaccines, treat-
ments, and vector control methods. For the populations currently at risk, cohort studies are
needed to determine both absolute and relative risks of pregnancies affected by asymptom-
atic and symptomatic Zika virus infection and the role of cofactors and effect modifiers, and
to define the full range of physical and developmental abnormalities that comprise the con-
genital Zika virus syndrome.
From Rapid Systematic Review to Living Systematic Review
Our systematic review deals with multiple neurological disorders and more detailed questions
about causality than other reviews. We reached the same conclusion as Rasmussen et al. [11],
but the larger number of studies allowed a more comprehensive and balanced summary of evi-
dence and of evidence gaps. In addition, our review addresses the association between Zika
virus and GBS. We also plan to examine other acute neurological disorders (S1 Text).
Our review will quickly become outdated because the pace of new publications is outstrip-
ping the time taken for the review process. The concept of a “living systematic review” has
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PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 17 / 27
been proposed as a way to combine rigour with timeliness for intervention research [150]
through the development of methods to incorporate new evidence as soon as it is available and
make evidence summaries available immediately. We are working on methods to produce a
living systematic review of the Zika causality framework that will incorporate new studies, pro-
vide frequent open access updates, and allow cumulative meta-analyses of both aggregate and
individual patient data from rigorous prospective studies as these become available. The decla-
ration by journal editors to improve access to data during public health emergencies [151, 152]
could be combined with the living systematic review approach to improve timeliness and open
access to research about causality [153].
In summary, rapid and systematic reviews with frequent updating and open dissemination
are now needed, both for appraisal of the evidence about Zika virus infection and for the next
public health threats that will emerge. This rapid systematic review found sufficient evidence
to conclude that Zika virus is a cause of congenital abnormalities and is a trigger of GBS.
Supporting Information
S1 PRISMA Checklist.
(PDF)
S1 Text. Background to assessment of causality in epidemiology, Zika causality framework
questions, supplementary methods, and results.
(PDF)
S2 Text. Epidemic curves of reported cases of dengue, chikungunya, Zika virus, and micro-
cephaly in Pernambuco state, Brazil, by epidemiological week, 2015 to 2016. From PAHO
Epidemiological Update 28 April 2016 (Fig 7, p7).
(PDF)
S1 Table. Bradford Hill’s “viewpoints” of causation, modifications by Gordis, and adapta-
tion to the dimensions of the causality framework, in order.
(PDF)
S2 Table. Zika causality framework for congenital abnormalities and GBS, according to
causality dimension.
(PDF)
S3 Table. Characteristics of included and excluded items, total 1,091 unique items.
(PDF)
S4 Table. Causality framework evidence for congenital brain abnormalities.
(PDF)
S5 Table. Quality assessment of the body of evidence about congenital brain abnormalities.
(PDF)
S6 Table. Causality framework evidence for GBS.
(PDF)
S7 Table. Quality assessment of the body of evidence about GBS.
(PDF)
S1 Fig. Flowchart of included and excluded items.
(TIF)
Zika Virus as a Cause of Neurological Disorders
PLOS Medicine | DOI:10.1371/journal.pmed.1002203 January 3, 2017 18 / 27
Acknowledgments
Expert panel members: Nahida Chakhtoura, National Institutes of Health/National Institute of
Child Health and Development, USA; Niklas Danielsson, European Centre for Disease Pre-
vention and Control, Sweden; Paul Garner, Liverpool School of Tropical Medicine, UK; Eva
Harris, Sustainable Sciences Institute, USA; Mauricio Hernandez Avila, Instituto Nacional de
Salud Publica, Mexico; Margaret Honein, Centers for Disease Control and Prevention, USA;
Bart Jacobs, Erasmus University, Netherlands; Thomas Janisch, University of Heidelberg, Ger-
many; Marcela Marıa Mercado Reyes, Instituto Nacional de Salud, Colombia; Ashraf Nabhan,
Ain Shams University, Egypt; Laura C. Rodrigues, London School of Hygiene and Tropical
Medicine, UK; Holger Schunemann, McMaster University, Canada; James Sejvar, Centers for
Disease Control and Prevention, USA; Tom Solomon, University of Liverpool, UK; Jan P.
Vandenbroucke, Leiden University Medical Centre, Netherlands; Vanessa Van der Linden,
Hospital Barao de Lucena, Brazil; Maria van Kerkhove, Institut Pasteur, France; Tatjana AvsičZupanc, University of Ljubljana, Slovenia.
WHO Zika causality working group members: Maria Almiron, Nathalie Broutet (chair),
Tarun Dua, Christopher Dye, Pierre Formenty, Florence Fouque, Metin Gulmezoglu, Edna
Kara, Anais Legand, Bernadette Murgue, Susan Norris, Olufemi T. Oladapo, William Perea
Caro, Pilar Ramon Pardo, Ludovic Reveiz Herault, and João Paulo Souza.
The views expressed in this article are those of the authors and do not necessarily represent
the decisions, policies, or views of the WHO or PAHO.
Author Contributions
Conceptualization: NL FK MR LR OTO NJB.
Data curation: FK MR AH.
Formal analysis: FK.
Funding acquisition: NL.
Investigation: NL FK MR LR RMV TVP AH.
Methodology: NL FK MR LR OTO NJB.
Project administration: FK NL NJB.
Supervision: NL NJB LR.
Visualization: FK MR NL.
Writing – original draft: FK NL LR OTO NJB.
Writing – review & editing: FK MR LR OTO TVP RMV AH NJB NL.
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